Project Summary/Abstract Biofilms are cohesive, multicellular microbial communities that are able to adhere to biotic or abiotic surfaces. The human microbiome contains numerous biofilm-forming bacterial species that help maintain normal human physiology. On the other hand, more than half of the 1.7 million hospital-acquired infections in the US are caused by biofilm-forming bacterial pathogens. The biofilm lifestyle is advantageous, because phenotypic diversity and coordination of cellular behaviors within biofilms provide bacterial populations with emergent capabilities beyond those of individual cells. For example, biofilms are orders of magnitude more tolerant towards physical, chemical, and biological stressors, most notably long-term treatments with antibiotic drugs or clearance attempts by the immune system. However, it remains largely unknown how such remarkable capabilities emerge from the behaviors of individual cells and the interactions between them. A critical barrier to rapid progress is the inability of conventional microscopes to resolve micrometer-sized bacterial cells in thick (>10 micrometers) biofilms in a non-invasive manner. The proposed research addresses this challenge by developing integrated experimental and computational technologies that enable non-invasive, 3D fluorescence imaging of pathogenic biofilms by lattice-light sheet microscopy, accurate single-cell segmentation and 3D shape measurements based on the acquired images, and simultaneous 3D tracking of thousands of cells inside biofilms. The ability to make single- cell measurements in dense microbial populations will enable researchers to correlate the spatial trajectory of each cell with that cells? gene expression and behavioral phenotype. Such information will provide an integrated understanding of how bacteria coordinate gene expression and social behaviors in 3D space and time. A fundamental understanding of biofilm biology will help inform new strategies for harnessing the emergent functional capabilities of microbial populations and for removing pathogenic biofilms from undesired environments.